The sarcoplasmic reticulum Ca2+ release channel (ryanodine receptor, RyR), plays an important role in Ca2+ homeostasis and signal transduction. There have been three different RyR isoforms (RyR1 "skeletal"; RyR2 "cardiac"; RyR3 "brain") described, but many of their structural properties as well as their functional roles in excitation- contraction (EC) coupling remain to be determined. I propose to take advantage of the fact that unlike mammalian muscle, non-mammalian muscle contains abundant quantities of the uncharacterized RyR3 isoform. I will first purify RyR3 from fish muscle, and secondly, compare its morphological characteristics and physiological regulation with the other RyRs. We hope to define distinct differences which reflect each isoform's role in EC coupling. Using molecular and immunological techniques we will also determine the tissue localization of RyR3 in order to discern if its expression is related to functional properties such as the frequency and/or the speed of contraction in muscles. Finally, since non-mammals contain muscles which express two RyR isoforms, and also contain muscles which express only one isoform, they provide an ideal system to investigate the functional significance of coexpression of RyRs in EC coupling. We propose to study sarcoplasmic reticulum preparations and single fibers from the two muscle populations to determine the role of coexpression in EC coupling.